Evaluation Of Cell Survival Research Articles

Evaluation of cell survival in different 3D-printed geometric shapes of human iPSC-derived engineered heart tissue.

Engineered Heart Tissue (EHT) is a promising tool to repair heart muscle defects and can additionally be used for drug testing. Due to the absence of an invitro vascularization, EHT geometry crucially impacts nutrient and oxygen supply by diffusion capacity. We analyzed cardiomyocyte survival in different EHT geometries. Different geometries with varying surface-area-to-volume-ratios were calculated (structure A (Ring) AS/V = 58.47 mm2/440 μL3, structure B (Infinity) 25.86 mm2/440 μL3). EHTs were generated from hiPSC-derived cardiomyocytes (4 × 106) and a fibrin/thrombin hydrogel. Cell viability was evaluated by RT-PCR, cytometric studies, and Bioluminescence imaging. Using 3D-printed casting molds, spontaneously beating EHTs can be generated in various geometric forms. At day 7, the RT-PCR analyses showed a significantly higher Troponin-T value in ring EHTs, compared to infinity EHTs. In cytometric studies, we evaluated 15% more Troponin-T positive cells in ring (73% ± 12%), compared to infinity EHTs (58% ± 11%, p = 0.04). BLI visualized significantly higher cell survival in ring EHTs (ROI = A: 1.14 × 106 p/s and B: 8.47 × 105 p/s, p < 0.001) compared to infinity EHTs during longitudinal cultivation process. Use of 3D-printing allows the creation of EHTs in all desired geometric shapes. The geometry with an optimized surface-area-to-volume-ratio (ring EHT) demonstrated a significantly higher cell survival measured by RT-PCR, Bioluminescence imaging, and cytometric studies using FACS analysis.

MECHANISM OF MICRORNA-218-5P IN MITOCHONDRIAL BIOGENESIS OF SEPSIS-INDUCED ACUTE KIDNEY INJURY BY THE REGULATION OF PGC-1Α.

Background: Sepsis-induced acute kidney injury (SI-AKI) is a kind of kidney dysfunction, which brings a lot of suffering. This study aimed to figure out the role of the miR-218-5p/PGC-1α axis in SI-AKI. Methods: AKI mouse model was established through cecal ligation and puncture. PGC-1α expression was activated using an activator ZLN005 before the serum and tissue samples were collected. Next, pathological structure and apoptosis of kidney tissues were observed. Levels of blood urea nitrogen, serum creatinine, and indicators of inflammation and oxidative stress were assessed. Moreover, reactive oxygen species and mitochondrial membrane potential levels, adenosine 5'-triphosphate content, and mitochondrial ultrastructure of kidney tissues were observed. HK2 cells were treated by lipopolysaccharide (LPS) to mimic sepsis in vitro , followed by evaluation of cell survival and apoptosis, inflammation, and oxidative stress. Subsequently, the binding relation between PGC-1α and miR-218-5p was predicted and validated. Then expression of PGC-1α and miR-218-5p was detected. PGC-1α and miR-218-5p expression were intervened to detect their influences in mitochondrial biogenesis. At last, miR-218-5p was overexpressed in ZLN005 (PGC-1α activating agent) pretreated SI-AKI mice to validate the mechanism. Results: PGC-1α is poorly expressed in SI-AKI, but overexpression of PGC-1α using ZLN005 alleviated SI-AKI injury and promoted mitochondrial biogenesis in AKI mice, and relieved LPS-induced cell injury. PGC-1α is a target of miR-218-5p. Downregulation of miR-218-5p expression in HK2 cells attenuated mitochondrial biogenesis disorder. Inhibition of PGC-1α annulled the role of miR-218-5p silencing in cells. In vivo , miR-218-5p overexpression partly reversed the protective role of ZLN005 in SI-AKI mice. Conclusion: miR-218-5p targeted PGC-1α to disrupt mitochondrial biogenesis, thereby exacerbating SI-AKI.

Equivalent Uniform Dose (EUD) and the Evaluation of Cell Survival in Spatially Fractionated Radiotherapy (SFRT)

SFRT has shown promise as a treatment modality to decrease normal tissue sparing without compromising tumor coverage, i.e., an increase in the therapeutic window compared to more conventional uniform radiation therapy (RT). The aim of this work is to examine and test several alternative bio-dosimetric parameters for the prediction of cell survival for normal-tissue and tumor cell lines irradiated in vitro with uniform and microbeam radiotherapy (MRT). A bespoke tungsten collimator with 50 parallel, 50 µm wide slits and 400 µm slit spacing was mounted into an x-ray cabinet. Human fibroblast (MRC5) and two human tumor cell lines (LN18 and A549) were irradiated with a range of doses (< 10 Gy) for uniform and MRT (50um slits, 400um center spacing) using kV X-rays. Average, mean and valley dose as useful predictive metrics of cell survival are compared to the equivalent uniform dose (EUD) with biological parameters estimated from uniform-dose experiments. We find that EUD, with linear-quadratic (LQ) model parameters, is more predictive for survival after SFRT than maximum, minimum or average dose. The maximum and average doses are correlated very poorly with in vitro cell survival. The difference in cell survival between uniform and MRT irradiation as a function of EUD is cell-type and dose dependent. The report results suggest that MRT is more effective at cell killing of tumor-cell lines than uniform irradiation for both tumor cell lines. However, MRT is less effective at killing normal tissue cells than uniform irradiation. EUD is a superior predictor of in vitro cell survival than other metrics sometimes used in the SFRT literature, including mean dose, maximum dose, and valley dose. The reported studies provide some evidence that SFRT may increase the therapeutic ratio by producing spatial dose distributions that effectively reduce normal-tissue damage with little or no change in biological damage to tumor cells. Additional studies are needed to further extend and generalize our results and to test our conclusions against a larger dose range, low and high linear energy transfer (LET) radiations and additional cell lines.

Evaluation of Viability and Cell Proliferation in Bone and Gingival on Dental Implant Fixtures with Active Sandblasted and Sandblasted Surfaces by the Cytotoxicity Test Method

In recent years, the technology of dental implants has advanced a lot and this has become one of the main reasons for dentists to refer to implants immediately after tooth extraction. Evaluation of cell survival in implantology studies to determine cell sensitivity determines the outcome of treatment. This study aimed to investigate the bone integration properties as well as the cytotoxicity between the implant surface and the jaw bone. In this study, cytotoxicity test was used to evaluate the toxicity and viability of sandblasted large grit acid etched, and sandblasted large grit acid etched active surfaces in 3A brand dental implants with G292 osteoblastic cells and human gingival fibroblasts cells are discussed. This operation was performed using a laboratory incubator of the German company MEMMERT for 24 hours, by neubauer lam cells counting for one hundred thousand cultured cells in each test at a temperature of 37 °C, a pressure of 1 atmosphere and 90% humidity. Based on the scanning electron microscopy images and the cytotoxicity test results, it can be seen that the bone graft of the implant, with the sandblasted large grit acid etched active surface treatment, is much better and also one week faster than the implant with the sandblasted large grit acid etched one. However, the viability of the implant with the sandblasted large grit acid etched active surface treatment for both G292 osteoblastic cells and human gingival fibroblasts cells samples was equal to 98.4% and 97.3%, respectively, and is lower than the sandblasted large grit acid etched surface treatment. The results show that the viability of the sandblasted large grit acid etched implant is about 1.5 to 2% higher than the sandblasted large grit acid etched active one, but the surface integrity of sandblasted large grit acid etched active is better than sandblasted large grit acid etched in all samples, and the treatment process is reduced by one week.

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses.

Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.

Mesenchymal stem cells and the neuronal microenvironment in the area of spinal cord injury.

Cell-based technologies are used as a therapeutic strategy in spinal cord injury (SCI). Mesenchymal stem cells (MSCs), which secrete various neurotrophic factors and cytokines, have immunomodulatory, anti-apoptotic and anti-inflammatory effects, modulate reactivity/phenotype of astrocytes and the microglia, thereby promoting neuroregeneration seem to be the most promising. The therapeutic effect of MSCs is due to a paracrine mechanism of their action, therefore the survival of MSCs and their secretory phenotype is of particular importance. Nevertheless, these data are not always reported in efficacy studies of MSC therapy in SCI. Here, we provide a review with summaries of preclinical trials data evaluating the efficacy of MSCs in animal models of SCI. Based on the data collected, we have tried (1) to establish the behavior of MSCs after transplantation in SCI with an evaluation of cell survival, migration potential, distribution in the area of injured and intact tissue and possible differentiation; (2) to determine the effects MSCs on neuronal microenvironment and correlate them with the efficacy of functional recovery in SCI; (3) to ascertain the conditions under which MSCs demonstrate their best survival and greatest efficacy.

204 MUSCLE REGENERATION AND TRACKING OF HUMAN MUSCLE PRECURSOR CELLS BY MRI IN VIVO

You have accessJournal of UrologyStem Cell Research1 Apr 2012204 MUSCLE REGENERATION AND TRACKING OF HUMAN MUSCLE PRECURSOR CELLS BY MRI IN VIVO Fahd Azzabi, Virginija Jovaisaite, Andreas Boss, Josiane Njiwa, Markus Rudin, Tullio Sulser, and Daniel Eberli Fahd AzzabiFahd Azzabi Zurich, Switzerland More articles by this author , Virginija JovaisaiteVirginija Jovaisaite Zurich, Switzerland More articles by this author , Andreas BossAndreas Boss Zurich, Switzerland More articles by this author , Josiane NjiwaJosiane Njiwa Zurich, Switzerland More articles by this author , Markus RudinMarkus Rudin Zurich, Switzerland More articles by this author , Tullio SulserTullio Sulser Zurich, Switzerland More articles by this author , and Daniel EberliDaniel Eberli Zurich, Switzerland More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.257AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Stress Urinary Incontinence (SUI), the involuntary loss of urine, is a medical problem that affects millions of people worldwide. Recent research suggests stem cell therapy as a potential solution to restore a normal sphincter function. Clinical trials to treat SUI by autologous Muscle Precursor Cells (MPC) transplantation are currently in preparation. In addition to functional follow-up, evaluation of cell survival and tissue formation is essential for understanding and improvement of cell therapies. Unfortunately, a biopsy of the newly engineered muscle would counteract the anticipated treatment. Therefore, tracking transplanted stem cell populations by a non-invasive manner, such as Magnetic Resonance Imaging (MRI), is desirable. One of the methods to render the cells clearly visible by MRI is intracellular incorporation of Superparamagnetic Iron Oxide Nanoparticles (SPION) prior to transplantation. In this study we explored detrimental effects of increasing intracellular levels of SPIO in vitro and aimed to define a safe concentration in which human MPCs can be easily detected by MRI, without altering their cellular functions. METHODS Human MPCs were harvested following standard protocols and then labelled with increasing concentration of SPION (Endorem®, 100–1600 ìg/mL). In the following passage labelling efficiency, cell viability, growth, molecular characteristics and differentiation were examined in vitro. MPCs labelled with 400 ìg/mL SPIO were then injected into the subcutaneous space of nude mice and followed for 4 weeks by MRI imaging. At harvest muscle tissue formation was assessed macroscopically and by immunohistochemistry. RESULTS Majority of cells were labelled, when 200 ìg/mL or more of SPIO was used. Labelling with more than 800ìg/mL of iron oxide reduced the viability of cells by 15-25%. In the long term, 800-1600 ìg/mL SPIO activated cell growth while it decreased MPC differentiation ratio by 10%. Using SPIO concentration of 400ìg/mL, no effects on cell viability, growth, differentiation and muscle-specific marker expression were detected, while labelling allowed the cells to be detected by MRI. Labelled human MPCs were then injected into the subcutaneous space of nude mice. Transplanted cells formed a muscle tissue, as confirmed by histological analysis. Location of the muscle was detectable by MRI for at least 4 weeks. CONCLUSIONS Our data concludes that the optimized conditions of MPC labelling can be safely used in clinics to track sphincter muscle regeneration in patients under SUI treatment by cell therapy. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e85 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Fahd Azzabi Zurich, Switzerland More articles by this author Virginija Jovaisaite Zurich, Switzerland More articles by this author Andreas Boss Zurich, Switzerland More articles by this author Josiane Njiwa Zurich, Switzerland More articles by this author Markus Rudin Zurich, Switzerland More articles by this author Tullio Sulser Zurich, Switzerland More articles by this author Daniel Eberli Zurich, Switzerland More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Pro-apoptotic effects of parathyroid hormone in intestinal cells

Apoptosis, a form of programmed cell death, is a process fundamental to normal growth and development, immune response, tissue remodeling after injury or insult, and homeostasis of the intestinal epithelium. Recently, it has become apparent that apoptosis is a crucial process in skeletal development and homeostasis, and that signaling by the parathyroid hormone (PTH) receptor can either promote or suppress apoptosis depending on the cellular context. In this study, we evaluated the role of PTH in intestinal apoptosis using human colonic Caco-2 cells. To that end, Caco-2 cells were exposed to PTH (10-8 mol/L) for 1-5 days. Evaluation of cell survival by use of resazurin staining, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) staining, and crystal violet staining revealed that PTH treatment diminishes the number of viable cells. Assessment of cells after PTH treatment by use of propidium iodide showed that the hormone increased the number of red-stained (dead) cells (178%, 5 days). Moreover, we found that the hormone induced disruption of actin filaments with changes to cellular shape, alteration of cell-to-cell junctions, externalization of membrane phosphatidylserine, chromatin condensation, and DNA fragmentation of the nucleus, which are morphological features consistent with apoptosis. In addition, PTH treatment revealed a cytosolic staining pattern of 14-3-3. However, the significance of such differential localization for 14-3-3 function remains unknown. Taken together, the present study suggests that PTH promotes apoptosis in Caco-2 intestinal cells.

VEGF-subtype specific protection of SCC and HUVECs from radiation induced cell death

The contribution of VEGF (vascular endothelial growth factor) to angiogenesis and tumor aggressiveness has been shown in several tumor types, but no comparative data regarding the impact of the VEGF-subtypes on endothelial and tumor cell protection are available. Therefore, we analysed the cytoprotective effects of the two major soluble VEGF-subtypes, VEGF-121 and -165, on HUVEC cell cultures (human umbilical vein endothelial cells) and squamous cell carcinoma (SCC) cell lines after ionizing radiation. We performed clonogenic analyses and proliferation assays for evaluation of cell survival and proliferative activity. Experiments were performed employing different intensities up to 8 Gy with or without added recombinant VEGF-121 or -165 and compared to non-irradiated cultures. To evaluate a possible contribution of the VEGF-receptors, we performed immunohistochemical stainings of VEGF-R1 (flt), -R2 (KDR,flk), and Neuropilin-1. In the SCC cell lines and HUVEC cultures, cell survival and proliferative activity were reduced in a dosage-dependent manner. The addition of VEGF-121 yielded a significant increase of resistance from 1.2 to 2.7 Gy (+125%) for killing 50% of subjected cells, while VEGF-165 was less effective in this regard (+83%). Conversely, in HUVEC cultures, 50%-survival was increased more strongly by VEGF-165 compared to VEGF-121 (+100% vs. +43%). Accordingly, proliferation was more intensely stimulated in HUVECs by VEGF-165 than by VEGF-121. VEGF-receptors were expressed in all cell cultures analysed at comparable levels. We conclude that the two VEGF-subtypes differentially increase the survival of tumor and endothelial cells after irradiation in vitro. We hypothesise, that the release of VEGF by the tumor protects tumor cells and endothelium resulting in increased radiation resistance.

Cyclin D1 antisense oligonucleotide inhibits cell growth stimulated by epidermal growth factor and induces apoptosis of gastric cancer cells.

The cyclin Dl protein is one of the cell cycle regulators required for cell cycle progression through Gl phase to S phase. The cyclin Dl‐cyclin‐dependent kinase (CDK) system is thought to control the cell cycle through mediating extracellular signals from mitogens, such as epidermal growth factor (EGF). In this study, we attempted to examine the therapeutic effect of cyclin Dl antisense oligonucleotides (AS/D1) on cell proliferation and apoptosis of the gastric cancer cell line MKN‐74, in the presence and absence of EGF‐stimulation. Evaluation of cell survival and DNA synthesis revealed that enhanced cell growth following EGF‐stimulation was completely inhibited by a 24 h pre‐incubation with 100 nM AS/D1. This inhibition was down to 19.3% compared with maximal DNA synthesis after stimulation with 3 nM EGF alone. Western blotting demonstrated that while EGF‐stimulation led to cyclin Dl over‐expression, AS/D1 inhibited cyclin Dl protein expression. We also demonstrated the induction of apoptosis in MKN‐74 cells by AS/D1. In conclusion, EGF‐stimulated MKN‐74 cell proliferation was inhibited by AS/D1, which could overcome EGF‐induced cyclin Dl over‐expression. AS/D1 also affected cell survival by inducing apoptosis through cell cycle arrest following cyclin Dl depletion. Thus, AS/D1 may be a candidate for use as a novel cancer therapy specifically targeted against the over‐expression of cyclin Dl enhanced by EGF in malignant cells

Evaluation of cell survival, DNA double strand breaks, and DNA synthesis during concurrent camptothecin and X-radiation treatments.

We evaluated cell survival, DNA double strand breaks (dsbs), and DNA synthesis following camptothecin (CPT) alone or concurrent CPT and X-radiation treatments in exponential-phase cultures of a radioresistant human melanoma cell line. Cell survival was measured by a clonogenic assay. DNA dsbs were measured by pulsed-field gel electrophoresis. DNA synthesis was measured by incorporation of (3)H-thymidine. We found that (i) concurrent CPT and X-radiation interacted additively, unlike previous results with plateau-phase cultures of these cells, which showed synergistic interaction; (ii) there were strong negative correlations (correlation coefficients of at least 0.82) between clonogenic surviving fractions and DNA dsbs following CPT alone or concurrent CPT and radiation treatments; and (iii) concurrent CPT and radiation (10 Gy) treatment did not completely inhibit DNA synthesis, even though addition of radiation to CPT did further decrease DNA synthesis (relative to CPT alone) at CPT concentrations below 20 microM. Our results suggest that during concurrent CPT and radiation treatment residual DNA dsb levels were good indicators of cell killing and that the absence of complete inhibition of DNA synthesis could at least in part explain the additive interaction between CPT and radiation.